Process for manufacture of in vivo stain composition

ABSTRACT

A process for synthesizing 2-amino-5-dimethlyaminophenyl thiosulfonic acid comprises the step of oxidizing N,N′-dimethyl-ρ-phenylene-diamine in the presence of a source of thiosulfate ions, while maintaining the temperature of the reaction mixture not higher than about 10° C. This compound is useful as an intermediate in the synthesis of toluidine blue O. A process for manufacturing toluidine blue O with improved yield, includes the step of preparing the intermediate 2-amino-5-diethylaminopropyl thiosulfonic acid according to the above described procedure.

This application is a continuation of my U.S.A. application Ser. No.09/110,788, filed Jul. 6, 1998, now U.S. Pat. No. 6,194,573 which was,in turn, a continuation-in-part of International Application, PCTUS/97/20981, filed Nov. 13, 1997, entitled IN VIVO STAIN COMPOSITION,PROCESS OF MANUFACTURE, AND METHODS OF USE TO IDENTIFY DYSPLASTICTISSUE.

This invention pertains to improved methods of manufacturing TBOcompositions, including the novel compositions which are disclosed in myabove-identified prior applications.

In another aspect, the invention concerns a method and process formanufacturing TBO compositions with improved yield, leading tomanufacturing economies and increased productive capacity of themanufacturing equipment.

The various embodiments of the invention and the practice thereof willbe apparent to those skilled in the art, from the following detaileddescription thereof, taken in conjunction with the claims and inconjunction with the drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a process flow diagram, depicting the improved process which Ihave discovered, for manufacturing TBO products, including the novel TBOproduct compositions disclosed in my above-identified priorInternational Application.

BACKGROUND OF THE INVENTION

In my prior International Application, I describe a novel generalprocess for manufacturing a TBO product and also a process formanufacturing a TBO composition which has specific chemicalcharacteristics, as required for regulatory approval for in vivoapplication to human tissue.

THE PRIOR ART

The classic synthesis of TEO, as practiced before the improvementsdisclosed in my above-identified International Application, isexemplified in the U.S. Pat. No. 418,055, issued Nov. 30, 1989, toDandliker et al. This synthesis is a series of three oxidation steps:(1) oxidation of N,N-dimethyl-ρ-phenylenediamine, e.g., with potassiumdichromate, to form 2-amino-5-dimethylaminophenyl thiosulfonic acid; (2)condensation of the thiosulfonic acid with o-toluidine, to form thecorresponding indamine-thiosulfonic acid; and (3) ring closure of theindamine-thiosulfonic acid, e.g., in the presence of zinc chloride atboiling temperature for about 30 minutes, to form TBO. The reactionmixture is then cooled and the TBO product of the ring-closure reactionis complexed and salted out, e.g., by treatment with sodium chloride andzinc chloride, to precipitate the TBO complex, e.g., as an TBO/ZnCl₂complex. Purification may be accomplished by repeated re-solution andre-precipitation, e.g., by re-solution in hot aqueous zinc chloridesolution and re-precipitation with sodium chloride/zinc chloride.

In my above-identified International Application, I disclosed a novelprocess in which the complexing agent is added to the reaction mixturebefore the ring closure (third) step of the Dandliker synthesis,preferably before the first oxidation step (Step 11, of FIG. 1) of theprocess. The process of my prior International Application was definedas an improvement of the Dandliker process, i.e., in a process whichincluded the steps of

oxidizing N,N-dimethyl-ρ-phenylene diamine ions in a first reactionmixture to form a first intermediate, 2-amino-5-dimethylaminophenylthiosulfonic acid,

oxidizing the first intermediate and condensation of the oxidizate witho-toluidine, in a second reaction mixture, to form a secondintermediate, indamine thiosulfonic acid,

oxidizing the second intermediate in a third reaction mixture to closethe indamine ring, forming a TBO reaction product, dissolved in thethird reaction mixture,

introducing a complexing reagent into said third reaction mixture, toform a TBO-complex product, and

separating said TBO-complex from said third reaction mixture,

the improvement on this prior art process comprising the step of addingthe complexing reagent at a stage earlier than the formation of thethird reaction mixture, preferably before the formation of the secondreaction mixture.

In the preferred practice of the process disclosed in my priorInternational Application, the conversion of the starting material,N,N′-dimethy-ρ-phenylene-diamine, to form a first intermediate,2-amino-5-dimethylaminophenyl thiosulfonic acid, was preferably carriedout at a temperature of below about 10° C., and most preferably at about5° C., for approximately 20 minutes, then warming the reaction mixtureto approximately 20-25° C., then adding the thiosulfonization agent (asource of thiosulfate ions, e.g., sodium thiosulfate pentahydrate), thenraising the temperature of the reaction mixture to an elevatedtemperature above room temperature, preferably to about 60° C. andcontinuing to stir the reaction mixture for approximately 30 minutes.

BRIEF DESCRIPTION OF THE INVENTION

I have now discovered that the yield of the TBO product prepared by theoverall process disclosed in my prior International Application issignificantly increased. The improved-yield process of the presentinvention is an improvement on the manufacturing process disclosed in myprior International Application, which includes the steps of

oxidizing N,N′-dimethyl-ρ-phenylene diamine in a first reaction mixture,to form a first intermediate, 2-amino-5-dimethylamino thiosulfonic acid,

oxidizing the first intermediate and condensing the oxidizate in asecond reaction mixture with o-toluidine, forming a second intermediate,indamine thio-sulfonic acid,

oxidizing the second intermediate to close the indamine ring thereof,forming a TBO-containing reaction product dissolved in a third reactionmixture,

introducing a complexing agent into a reaction mixture before theformation of the third reaction mixture, to form a TBO-complex product,precipitating the TBO-complex product from the third reaction mixtureand separating the TBO-complex product from the precipitate,

The improvement of the present invention, for increasing the yield ofTBO-complex product from the process of the present application,comprises the steps of introducing a source of thiosulfonate ions intothe first reaction mixture to form the first intermediate, whilemaintaining the reaction mixture at a reduced temperature of about 10°C. and continuing the reaction at this reduced temperature beforeraising the temperature to an elevated temperature.

I have also discovered an improved process for manufacturing indaminethiosulfonic acid which comprises the steps of oxidizingN,N′-dimethyl-ρ-phenylene diamine in a reaction mixture containing itand an oxidizing agent in a reaction mixture and introducing a source ofthiosulfate ions into the reaction mixture, maintaining the temperatureof the oxidation-thiosulfonization reaction mixture at or below about10° C. for approximately 30 minutes, and raising the temperature to anelevated temperature for approximately 30 minutes, to form a solution ofindamine thiosulfonic acid dissolved in the reaction mixture.

DETAILED DESCRIPTION OF THE INVENTION

In the improved-yield process of the present invention, thethiosulfonization agent is introduced into the first oxidation reactionmixture at a reduced temperature of from about 5-10° C. and the reactionis continued at that reduced temperature before warming the reactionmixture to an elevated temperature of about 60° C., rather than warmingthe first reaction mixture to approximately 20-25° C. before introducingthe thiosulfonization agent and then warming the resultingoxidization-thiosulfonization reaction mixture to the elevatedtemperature. After completing the thiosulfonization, the manufacturingprocess is continued in accordance the procedures disclosed in my priorInternational Application, i.e., the intermediate,2-amino-5-dimethylaminophenyl thiosulfonic acid, is further oxidized andcondensed with o-toluidine, in a second reaction mixture, forming asecond intermediate, indamine thiosulfonic acid, the second intermediateis further oxidized in a third reaction mixture to close the indaminering, forming a TBO reaction product, complexing the TBO reactionproduct in the third reaction mixture, forming a TRO-complex product,and separating the TBO-complex from the third reaction mixture.

Thus, in accordance with my improved-yield process, the timing ofaddition of the thiosulfonization agent to the first reaction mixture ischanged so that it is added before the temperature of the first reactionmixture is raised in later processing steps.

Formation of First Reaction Mixture

Referring to FIG. 1, an aqueous solution of the starting material 10 isoxidized 11, preferably at less than 10° C., especially at about 5° C.,by reaction with a suitable oxidizing agent 12, e.g., potassiumdichromate 12, in the presence of acid, aluminum sulfate and a reagent,13 (which is believed to complex the intermediates) and is used in alater stage of the process to complex the TBO composition components),e.g., zinc chloride. Then, before raising the temperature of the firstreaction mixture, a source of thiosulfate ions 14, e.g., sodiumthiosulfate, is added and the reaction is continued at such reducedtemperature for approximately 30 minutes and later at an elevatedtemperature of about 60° C., to form a first reaction mixture 15containing the first intermediate, 2-amino-5-dimethylaminophenylthiosulfonic acid.

Formation of Second Reaction Mixture

The first reaction mixture 15 is then further reacted, preferably at atemperature of not greater than about 10° C., with additional oxidizingagent 16, e.g., potassium dichromate, and o-toluidine hydrochloride 17,in a condensation step 18 to form the second intermediate, acondensation product, indamine thiosulfonic acid in the second reactionmixture 19.

Formation of Third Reaction Mixture

The second reaction mixture 19 is then further oxidized 21, preferablyby addition of a suitable oxidizing agent 22, e.g., potassiumdichromate, at a temperature of not greater than about 10° C. This isfollowed by the addition of copper sulfate, zinc chloride complexingagent, acid and heating to 100° C. to effect closure of the indaminering, forming TBO in a third reaction mixture 24. At this point the TBOis separated from the third reaction mixture and purified.

Separation/Purification of TBO

For example, in the presently preferred embodiment of the process of thepresent invention, the TBO is precipitated from the third reactionmixture by complexation of 24 with a suitable complexing agent 25, e.g.,zinc chloride, to form the complex TBO-zinc chloride double salt. Theprecipitate is filtered 26 from the liquid phase and washed with sodiumchloride solution 27. The washed filter cake is then redissolved 28 in acritical¹ volume of water 29 to form a TBO solution 30, which is thenfiltered 31 to remove undissolved solids 32 a, which are discarded. ZincChloride, followed by a critical² volume/concentration of sodiumchloride 33 is then added to the filtrate 32 to again precipitate theTBO-zinc chloride double salt. Then, the TBO-zinc chloride double saltis separated from the mixture by filtration, to yield a TBO-zincchloride/TBO hydrochloride filter cake 34.

¹ If too much water is used it prevents isolation of the TBO. If toolittle water is used (1) all of the TBO does not get dissolved, reducingthe yield and (2) it decreases the purity of the product.

² If too little sodium chloride is used, all of the product will not besalted out, reducing yield. If too much sodium chloride is used it willcause impurities to precipitate out along with the TBO, decreasing thepurity of the product.

As indicated by the dashed line 35, the TBO filter cake 34 can beredissolved, filtered, re-precipitated and re-isolated multiple times toachieve the desired degree of purity and yield of TBO. The finalpurified filter cake complex product 34 is then dried 35, e.g., inconventional convection oven and/or vacuum oven and the dried filtercake 36 is ground and blended 37 to yield the final TEO product 38. Thefinal TBO product contains both the zinc chloride double-salt of TBO(Formula X) and the chloride salt of TBO (Formulas I & II).

WORKING EXAMPLE

The following example is presented to illustrate the practice of theinvention in such terms as to enable those skilled in the art to makeand use the novel TBO compositions, to practice the novel diagnosticmethods using such TBO compositions and to practice the novel processfor preparing TBO compositions, which together form the variousembodiments of the invention, and to indicate to those skilled in theart the presently known best modes for practicing the variousembodiments of the invention. These examples are presented asillustrative only and not as indicating limits on the scope of theinvention, which is defined only by the appended claims.

Example 1 Manufacturing Process

This example illustrates, in the detail required to satisfy regulatoryrequired GMP conditions, the exact procedures for carrying out thecommercial scale manufacture of a batch of TBO dye product, according tothe process which embodies the presently known best mode of theinvention.

Preparation of Raw Materials Solutions

Equipment/Supplies:

A. Ohaus IP15KS Balance

B. AnD HV150KAI Balance

C. Fairbanks H90-5150 Balance

D. OHAUS WB25/1-20W Balance

E. Cole Parmer (51201-30) and Thermolyne (S25535)Stirrers

F. Sampling devices, such as steel scoops, drum samplers, etc.

G. Erlyenmeyer flasks, beakers, carboys and other appropriate glassware.

H. Production Solution Labels.

Safety:

Protective equipment, such as gloves, safety glasses, lab coats, andrespirators should be worn when handling chemicals according to MSDSguidelines.

Raw Material Solutions Preparation Procedure:

To Hydrochloric Acid, 1364.2 g (±5.5 g) add 1364.2 g (±5.5 g) of USPPurified water. Stir until the solution is clear.

To Aluminum Sulfate Hexadecahydrate, 1779.1 g (±7.0 g) add 2548.9 g(±10.0 g) of USP Purified water. Stir until the solution is clear.

To Zinc Chloride, 7384.6 g (±30.0 g), add 2786.7 g (±11.0 g) of USPPurified water. Stir until the solution is clear.

To Potassium Dichromate, 2101.9 g (±8.0 g), add 25203.8 g (±100 g) ofUSP Purified water. Stir until the solution is clear.

To Sodium Thiosulfate Pentahydrate, 1526.6 g (±6.0 g), add 2043.6 g(±8.0 g) of USP Purified water. Stir until the solution is clear.

To Copper Sulfate Pentahydrate, 509.7 g (±2.0 g), add 1613.1 g (±6.0 g)of USP Purified water. Stir until the solution is clear.

To Sulfuric Acid, 600.0 g (±2.0 g), add 600.0 g (±2.0 g) of USP Purifiedwater. Stir until the solution is clear.

To Sodium Chloride, 70.4 kg (±250 g), add 234.4 kg (±850 g) of USPPurified water. Stir until the solution is clear.

SAFETY

Protective equipment, such as gloves, safety glasses, lab coats, andrespirators should be worn when handling chemicals according to MSDSguidelines.

SYNTHESIS

Synthesis Equipment and Supplies:

LFE Control Panel (3000)

gallon Jacketed Glass Lined Purification Tanks with lid (E71224)

Two 100 gallon Jacketed Glass Lined Purification Tank with lids (P1,PT-001)(P2, L-13621)

FTS Recirculating Cooler (RC96C032) and 500 gallon Cold Storage Tank(500CST)

Three Caframo Mixers (BDC-1850) (R1, 18500961)(P1, 18501148) (P2,18501173) with shaft and impeller

Lightning Mixer (L1U08) (201550)

Three Heat Exchangers (Gardner Machinery) (R1, 01960763) (P1, 01960764)(P2, 08950727)

Three 12KW Jacket Fluid recirculators (Watlow, BLC726C3S 20)

Three Recirculation Pumps (Sta-Rite, JBHD-62S, C48J2EC15)

Masterflex Digital Peristaltic Pump (A94002806)

Masterflex Peristaltic Pump (L95003320)

Cole Parmer Peristaltic Pump (B96002074)

Neutsche Filtration unit (70-2038, 43421-1)

Two Buchner Filtration Units (Z11,624-6, Z10,441-8)

Siemens Vacuum Pump (F2BV2)

60 Gallon Glass Lined Collection Tank with lid (86854, E164-1186)

Air Compressor (DF412-2) (9502312538)

Flow Controller (3-5500) (69705069190)

Six Batch Controllers (3-5600) (#1,69705069191, #2, 69705069199, #3,69705069194, #4, 69705058829, #5, 69705058805, #6, 69705069195)

Six Flow Sensors (#1, 69704295165, #2, 69704024995, #3, 69704024994, #4,69704025027, #5, 69612178606, #6, 69703120990)

Four Diaphragm Pumps (M1)

Four Surge Suppressers (A301H) (#2, 15557, #3, 15561, #4, 15558, #5,15559)

Four Air Regulators (CFR10)

Four Solenoid Valves (used with air regulators)

Four Low Flow Sensors (FS-500)

Three Solenoid Valves (EASM5V16W20)

Air Filter/Regulator (T1R)PTFE / F06R113AC

Filter media, Polypropylene (7211-1)

Filter media, Whatman Grade 52

PharMed tubing (−18, −82, −90)

pH Meter; Hanna 9321 (1303675) & Orion 620 (001911)

Spectrophotometer 20 (3MU7202070)

Fisher Scientific Vacuum Oven (9502-033)

VWR 1370 FM forced air oven (1370FM)

Dust/Mist Respirator

Thomas Wiley Laboratory Mill (3375-E10)

Patterson-Kelley Blender (Blendmaster, C416578)

OHAUS TS4KD Balance

OHAUS IP15KS Balance

Mettler AG 104 Balance

AnD HV150KA1 Balance

Fairbanks H90-5150 Balance

OHAUS AS123 Printer

OHAUS AS142 Printer

AD-8121 Multifunction Printer

Citizen iDP 3540 Dot Matrix Printer

Hewlett Packard HPLC (1050)

Ultrasonic Cleaner (8892-DTH, QCC9601 005C)

Type K Thermocouple Temperature Recorder (KTx, 6292753, 6355146)

Erlenmeyer Flasks (8L, 6 L, 4 L, 1 L)

Beakers (8L, 6L, 500 mL, 250 mL)

Carboys (4L, 10L, 50 L)

HDPE Drums (55 gallon, 100 gallon)

Volumetric Flasks (100 mL)

Plastic Funnel

Pastuer Pipettes & Bulbs and Volumetric Pipettes (10 mL,

5 mL) & Bulb

Bellows (25 mL, 50 mL)

Weigh Paper

Spatulas

Packaging Material (containers, lids, labels)

Raw Material Solutions

SYNTHESIS: Step 1

Synthesis of 2-Amino-5-Dimethylaminophenyl thiosulfonic Acid

Check the integrity of the USP water system. To the reactor add theweighed USP Grade Purified Water (28,000 g±100.0 g) and stir at 190±10RPM. Record the conductivity of the USP water at the time the water wasdispensed.

Add N,N-dimethyl-1,4-phenylenediamine (5.128 mol. 720.0 g±3.0 g). Thematerial should be added as a powder (no lumps). Stir 10 minutes (±5minutes).

Add hydrochloric acid (6 N, 1136.9 g±5.0 g). Stir 15 minutes (±5minutes).

Ensure the pH meter is calibrated according to SOP # LM-007. Take areaction mixture sample of approximately 10 mL using a plastic samplingdevice. Mark the sample lot #.IPS1a. Check the pH and record. The pHmust be 2.8-3.8 @ 25° C.±5° C.

Add aluminum sulfate hexadecahydrate solution (4328.0 g ±21.0 g). Stir10 minutes (±5 minutes) at 275±10 RPM.

Add zinc chloride solution (3641.5 g±18.0 g). Cool to 4° C.±1° C.

Once the temperature (PV1) is 4° C.±1° C. add potassium dichromatesolution (6532.4 g±32.0 g) over a 20 minute period (±5 minutes). Whenaddition is complete stir 20 minutes (±5 minutes) and then change theSet Point (SP1) to 25.0° C. from the Main Menu.

While maintaining the temperature at less than 10° C., add sodiumthiosulfate pentahydrate solution (3570.2 g±18.0 g). Stir the solutionat ˜5° C. for 30 minutes (±5 minutes).

Change the Set Point to 25° C. When the temperature (PV1) reaches 25°C.±5° C., stir for 20 minutes. Change the Set Point on the LFEcontroller to 10.0.

Once the temperature has reached 13.0° C.±2.0° C. take a reactionmixture sample of approximately 10 mL using a plastic sampling device.Mark the sample lot #.IPS1b. Check the pH and record. The pH must be3.1-4.1 @ 25° C.±5° C.

SYNTHESIS: Step 2

Synthesis of Indamine Thiosulfonic Acid

Weigh out o-toluidine (604.4 g±2.5 g) and cool to 18° C.±3° C. in an icebath. Add hydrochloric acid (6 N, 1230.7 g±5.0 g) to the o-toluidineslowly. Remove the o-toluidine hydrochloride from the ice bath and allowthe solution to cool to 38° C.±30° C. Add the solution to the reactionmixture and stir 5 minutes (±3 minutes).

Add potassium dichromate solution (6532.4 g±32.0 g) over a 20 minuteperiod (±5 minutes). When addition is complete stir 10 minutes (±5minutes).

Change the controller Set Point (SP1) to 60.0. Once the reaction mixturetemperature reaches 60.0° C.±3° C. allow the mixture to stir 25 minutes(±5 minutes). A precipitate will form consisting of a green indamine.

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS2. Record the solution color.

SYNTHESIS: Step 3

Synthesis of Toluidine Blue O and Toluidine Blue O Zinc Chloride DoubleSalt:

Set the LFE controller Set Point to 7.0. Once the reaction mixturetemperature reaches 10.0° C.±3° C. add potassium dichromate solution(6532.4 g±32.0 g) over a 20 minute period (±5 minutes). When addition iscomplete stir 20 minutes.

Add potassium dichromate solution (5225.9 g±26.0 g) over a 20 minuteperiod (±5 minutes). When addition is complete stir 20 minutes (±5minutes).

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS3.

Add zinc chloride solution (3641.5 g±18.0 g). Stir 20 minutes (±5minutes) at 350±10 RPM.

Add copper sulfate pentahydrate (2122.8 g±10.0 g). Stir 15 minutes (±5minutes).

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS4.

Change the controller Set Point (SP1) to 100.0. Once the reactionmixture temperature reaches 67.0° C.±3° C. begin to add sulfuric acidsolution to pH 2.9±0.3 by adding aliquots (500 mL, 250, 125 mL, etc.).Stir for 5 to 10 minutes after each addition and check pH.

Once the reaction mixture temperature reaches 100.0° C.±3° C. allow themixture to stir 35±5 minutes.

Change the controller Set Point (SP1) to 35.0. Once the reaction mixturetemperature reaches 70.0° C.±3° C. take a reaction mixture sample ofapproximately 10 mL using a pipette. Mark the sample lot #.IPS5.

Change the controller Set Point (SP1) to 2.5. Cool to 2.5° C. in 4 hoursand Hold at 2.5° C.±2.0° C. for 4 to 18 hours.

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS6. Record the solution color. Check the pH andrecord. Filter the sample through 0.45 micron filter paper. Takeapproximately 100 milligrams of the precipitate and dissolve inapproximately 100 mL of HPLC water. Filter the solution through 0.45micron filter paper. Label the solution Lot # .IPS7 and analyze thesample by the RP-HPLC Toluidine Blue O Analysis Method. See Example 3.Record the results.

Purification: Step 1

Filter the reaction mixture through suitable filter media (Whatman Grade52).

When the reactor is empty weigh out 24.0 kg±150.0 g of 30% NaCl solutionand add 24.0 kg±150.0 g of USP water (record conductivity of thedispensed water). Close the reactor bottom valve and add the 15% NaClsolution to the reactor. Stir the solution briefly. When the filtrationis complete add the NaCl solution to the filtration unit to rinse thefilter cake. Collect the filtrate into the same container and Labellot#.HW1 (hazardous waste 1).

Process filtrate (lot#. HW1) according to waste disposal procedures.

Check the 100 gallon glass lined, jacketed purification tank # 1condition and make certain the tank has been properly labeled as CLEANEDwith date and signature. Equip the tank with a HDPE lid, Caframostirrer, stir shaft, propeller and thermocouple probe inserted into aplastic thermocouple well. Check that the bottom valve is off and theoutlet is capped.

Label the Tank with Lot#.P1A (Purification 1A).

Weigh out 190.0 kg±1.0 kg of USP water into a HDPE container (recordconductivity of the dispensed water) and transfer the water toPurification Tank 1. Stir the mixture at 350 RPM. Once the NaCl wash ofthe filter cake is complete add the filter cake to Purification Tank 1while stirring.

Stir the mixture 2 to 4 hours. Take a sample (through the bottom valve)of approximately 50 mL. Mark the sample lot#.IPS8. Record the solutioncolor.

Set the Purification Tank 1 LFE controller to 75.0 (SP1).

When the mixture temperature (PV1) reaches 75.0° C.±3° C. change the SetPoint on the controller to 40.0.

Allow the mixture to stir at 40° C. and 350 RPM for 12 to 36 hours.

Take a sample (through the bottom valve) of approximately 50 mL. Markthe sample lot#.IPS9. Record the solution color. Check the pH andrecord. Measure 1.0 mL of the sample with a 1.0 mL pipette and dilute to100 mL in a volumetric 100 mL flask. Label the sample lot#.IPS9A. Thentake 10.0 mL of this solution with a 10.0 mL pipette and dilute to 100mL in a volumetric 100 mL flask. Label the sample lot#.IPS9B. Measurethe absorbance of these samples using the spectronic 20±. Record theresults. The absorbance of sample 9B should be ≧0.220.

Purification: Step 2

Filter the mixture through filter media in the filtration unit. Collectthe filtrate into a Tared HDPE container with lid.

Check the 100 gallon glass lined, jacketed purification tank # 2condition and make certain the tank has been properly labeled as CLEANEDwith date and signature. Equip the tank with a HDPE lid, Caframostirrer, stir shaft, propeller and thermocouple probe inserted into aplastic thermocouple well. Check that the bottom valve is labeled asCLEANED, off (horizontal position) and the outlet is capped.

Label the Tank with Lot#.P2A (Purification 2A), date and signature.

When the filtration is complete weigh the container and solution.Subtract the tare weight. Record the solution weight. Calculate thesolution volume.

(TBO soln wt. g)(100.0 mL TBO soln/100.42 g TBO soln)=ml of TBO soln

Label the filter cake lot#.HW2 (Hazardous Waste 2) and process accordingto waste disposal procedures.

Into a clean HDPE container weigh out a quantity of 30% NaCl solutionequal to the solution volume recorded above using the following formula:

(mL of TBO soln)(116.91 g NaCl soln/100.0 mL NaCl soln)=g of NaCl soln

Sample 10 mL of the filtrate and check the pH. Label lot#.IPS10. The pHmust be 3.0-4.0. Transfer the filtrate (by weight) to Purification Tank2. Stir the solution at 350 RPM.

Add zinc chloride solution (1636.3 g±6.5 g)

Transfer the NaC1 solution (by weight) to Purification Tank 2.

Set the Purification Tank 2 LFE controller to 75.0 (SP1).

When the mixture temperature (PV1) reaches 75.0° C.±3° C. change the SetPoint on the controller to 5.0.

Cool to 5° C. in 6 hours and Hold at 50° C.±4.0° C. for 4 to 24 hours.

Take a sample (through the bottom valve) of approximately 50 mL. Markthe sample lot#.IPS11.PT2.

PROCESSING

i. Filter

Filter the mixture through tared filtration media (Whatman Grade 52) inthe filtration unit

Weigh out 12 kg±50 g of 30% sodium chloride solution and dilute with 12kg±50 g of USP water (record conductivity of the dispensed water). Washthe filter cake with the 15% sodium chloride solution by adding thesolution directly to the buchner. When the filtration is completecarefully remove the filter paper containing the toluidine blue Oproduct.

Process Lot#.HW3 (Hazardous Waste 3) according to waste disposalprocedures.

ii. Dry

Place the TBO product in the oven and dry at 50.0° C.±3.0° C. for 5±1hours. Label the oven lot#.PRE-DRY.

Remove the product from the forced air oven and place in the VacuumOven. Dry at 45.0° C.±3.0° C. @ 28 Hg±2 Hg for 10±2 hours. Label theoven lot#.DRY.

iii. Weigh

Remove the product and weigh the Toluidine Blue O and filter. Subtractthe filter weight and record the TBO weight.

Using a stainless steel spatula carefully remove the product from thefilter paper. Wear a Dust/Mist respirator. Weigh the Toluidine Blue.

iv. Grind

Transfer the product to the TOLUIDINE BLUE O FINISHING AREA. Check theThomas Wiley Laboratory Mill condition and make certain the mill hasbeen properly labeled as CLEANED with date and signature. Use the 0.5 mmscreen. Attach a clean container to the delivery chute. The chamber doormust be closed and latched.

Close the sliding shutter at the bottom of the hopper, remove the hopperlid and add the sample. Replace the hopper lid. Turn the mill ON andopen the sliding shutter slightly. Feed sample into the mill chamberslowly enough so that the mill does not slow down or become jammed.

Once the grinding is complete carefully remove the mason jar from thedelivery chute.

v. Blend

Check the Patterson-Kelly Lab Blender condition and make certain theblender has been properly labeled as CLEANED with date and signature.

Transfer the Toluidine Blue O product to the blender container and closethe lid. Set the timer to 15 minutes

vi. Test

Sample the product for testing. Analyze the sample by the RP-HPLCToluidine Blue O Analysis Method. Record the results.

Having described my invention in such terms as to enable persons skilledin the art to understand the invention and practice it, and, havingidentified the presently preferred embodiments thereof, I claim:

What is claimed is:
 1. In a process for manufacturing toluidine blue O(“TBO”), which process includes the steps of: oxidizingN,N′-dimethly-ρ-phenylene diamine in a first reaction mixture,introducing a source of thiosulfate ions into said first reactionmixture, to form a first intermediate, 2-amino-5-dimethylaminophenylthiosulfonic acid, further oxidizing and condensing said firstintermediate with o-toluidine, to form a second intermediate,indamine-thiosulfonic acid, further oxidizing said second intermediateto close the indainine ring thereof, to form a TBO-containing reactionproduct in a third reaction mixture, and separating the TBO-containingreaction product from the third reaction mixture, the improvementcomprising the step of adding potassium dichromate as the oxidationagent of N,N-dimethyl-ρ-phenylenediamine to form2-amino-5-dimethylaminophenyl thiosulfonic acid.
 2. In a process formanufacturing toluidine blue O (“TBO”), which process includes the stepsof: oxidizing N,N′-dimethly-ρ-phenylene diamine in a first reactionmixture, introducing a source of thiosulfate ions into said firstreaction mixture, to form a first intermediate,2-amino-5-dimethylaminophenyl thiosulfonic acid, further oxidizing andcondensing said first intermediate with o-toluidine, to form a secondintermediate, indamine-thiosulfonic acid, further oxidizing said secondintermediate to close the indamine ring thereof, to form aTBO-containing reaction product in a third reaction mixture, andseparating the TBO-containing reaction product from the third reactionmixture, the improvement comprising the step of adding sodiumthiosulfate pentahydrate as the thiosulfonization agent before furtheroxidizing and condensing said first intermediate with o-toluidine, toform a second intermediate, indamine-thiosulfonic acid.
 3. In a processfor manufacturing toluidine blue O (“TBO”), which process includes thesteps of: oxidizing N,N′-dimethly-ρ-phenylene diamine in a firstreaction mixture, introducing a source of thiosulfate ions into saidfirst reaction mixture, to form a first intermediate,2-amino-5-dimethylaminophenyl thiosulfonic acid, further oxidizing andcondensing said first intermediate with o-toluidine, to form a secondintermediate, indamine-thiosulfonic acid, further oxidizing said secondintermediate to close the indamine ring thereof, to form aTBO-containing reaction product in a third reaction mixture, andseparating the TBO-containing reaction product from the third reactionmixture, the improvement comprising the step of adding zinc chloride tofacilitate ring closure of the indamine-thiosulfonic acid and to formTBO.
 4. In a process for manufacturing toluidine blue O (“TBO”), whichprocess includes the steps of: oxidizing N,N′-dimethly-ρ-phenylenediamine in a first reaction mixture, introducing a source of thiosulfateions into said first reaction mixture, to form a first intermediate,2-amino-5-dimethylaminophenyl thiosulfonic acid, further oxidizing andcondensing said first intermediate with o-toluidine, to form a secondintermediate, indamine-thiosulfonic acid, further oxidizing said secondintermediate to close the indamine ring thereof, to form aTBO-containing reaction product in a third reaction mixture, andseparating the TBO-containing reaction product from the third reactionmixture, the improvement comprising the steps of: (a.) adding potassiumdichromate as the oxidation agent of N,N-dimethyl-ρ-phenylenediamine toform 2-amino-5-dimethylaminophenyl thiosulfonic acid; (b.) adding sodiumthiosulfate pentahydrate as the thiosulfonization agent before furtheroxidizing and condensing said first intermediate with o-toluidine, toform a second intermediate, indamine-thiosulfonic acid; and (c.) addingzinc chloride to facilitate ring closure of the indamine-thiosulfonicacid and to form TBO.
 5. In a process for manufacturing toluidine blue O(“TBO”), which process includes the steps of: oxidizingN,N′-dimethly-ρ-phenylene diamine in a first reaction mixture,introducing a source of thiosulfate ions into said first reactionmixture, to form a first intermediate, 2-amino-5-dimethylaminophenylthiosulfonic acid, further oxidizing and condensing said firstintermediate with o-toluidine, to form a second intermediate,indamine-thiosulfonic acid, further oxidizing said second intermediateto close the indamine ring thereof, to form a TBO-containing reactionproduct in a third reaction mixture, and separating the TBO-containingreaction product from the third reaction mixture, the improvementcomprising at least one step selected from the group consisting of thefollowing steps: (a.) adding potassium dichromate as the oxidation agentof N,N-dimethyl-ρ-phenylenediamine to form 2-amino-5-dimethylaminophenylthiosulfonic acid; (b.) adding sodium thiosulfate pentahydrate as thethiosulfonization agent before further oxidizing and condensing saidfirst intermediate with o-toluidine, to form a second intermediate,indamine-thiosulfonic acid; and (c.) adding zinc chloride to facilitatering closure of the indamine-thiosulfonic acid and to form TBO.